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The role of MTH1 in ultraviolet radiation-induced mutagenesis
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ultraviolet radiation (UVR) is known to be highly mutagenic. What types of DNA lesions that are induced by different UVR wavelengths are still a matter of debate. UVR induces mutagenesis mostly by the formation of photoproducts and the induction of reactive oxygen species (ROS). ROS can give rise to mutations via oxidation of nucleotides in the DNA or the nucleotide pool. Oxidized nucleotides in the nucleotide pool can thereby be incorporated into the DNA during replication and ultimately give rise to mutations. MTH1 however, dephosphorylates oxidized nucleotides in the nucleotide pool, in particular 8-oxo-dGTP and 2-OH-dATP, and inhibits their incorporation into the DNA.The aim of the present study was to investigate the role of MTH1 in mutagenesis and cytogenetic damage induced by UVR in a human lymphoblastoid TK6 cell line. The clonogenic survival, mutant frequency and micronucleus frequency were measured following exposure to UVA, UVB and UVC in MTH1-knockdown and wild-type TK6 cells. As a biomarker for oxidative damage the level of intracellular and extracellular 8-oxo-dG was measured in TK6 cells exposed to UVA. The mutational spectra of UVA-induced mutations at the thymidine kinase gene in MTH1-knockdown and wild-type TK6 cells were investigated.The results show that MTH1 protects against UVA and UVB mutagenesis significantly. MTH1, however, has been shown to offer no protection against UVR-induced cytogenetic damage and is therefore suggested to mainly inhibit mutagenesis. The mutational spectra show that GC>AT and AT>GC transitions are the dominant mutation types in cells exposed to UVA.In conclusion, MTH1 protects TK6 cells against mutagenesis induced by longer wavelengths of UVR. This indicates that the nucleotide pool is a significant target in mutagenesis for longer wavelengths of UVR. The type of mutations induced by UVA, GC>AT and AT>GC, can be formed by the incorporation of 2-OH-dATP from nucleotide pool into the DNA. UVA is therefore suggested to induce mutations by induction of oxidized nucleotides such as 2-OH-dATP.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2015. , 46 p.
Keyword [en]
ultraviolet radiation, MTH1, reactive oxygen species, 8-oxo-dGTP, 2-OH-dATP, micronucleus
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-113320ISBN: 978-91-7649-096-9 (print)OAI: oai:DiVA.org:su-113320DiVA: diva2:784070
Public defence
2015-03-06, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2015-02-12 Created: 2015-01-28 Last updated: 2016-11-02Bibliographically approved
List of papers
1. Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA
Open this publication in new window or tab >>Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA
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2011 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 715, no 1-2, 13-18 p.Article in journal (Refereed) Published
Abstract [en]

UVA has been suggested to play an important role in UV-induced mutagenesis. The mechanisms by which UVA induces mutations are still a matter of debate. Our aim was to investigate the protective capacity of hMTH1, a nucleotide pool sanitization enzyme with 8-oxodGTPase activity. Human B lymphoblastoid cells were stably transfected with shRNA directed against hMTH1. Clonogenic survival, mutations, intracellular and extracellular levels of 8-oxodG (8-oxo-7, 8-dihydro-2'-deoxyguanosine) and dG in the nucleotide pool of UVA-irradiated transfected and non-transfected cells were investigated. Mutations were determined in the thymidine kinase locus. Intracellular 8-oxodG and dG were measured using a modified ELISA and HPLC, respectively, after extraction of the nucleotide pool and conversion of nucleotides to their corresponding nucleosides. 8-oxodG in the medium was measured using ELISA. UVA-induced mutations were significantly higher while the survival was slightly lower in transfected compared to non-transfected cells. The increased mutation rate in transfected cells at increased exposure correlated with enhanced levels of 8-oxodG in the nucleotide pool, and a somewhat reduced level of 8-oxodG in the medium. The results indicate that the nucleotide pool is a significant target for UVA-induced mutations and implicates that hMTH1 plays an important role in protecting cells from UVA-induced oxidative stress.

Keyword
Oxidative stress, hMTH1, Mutagenesis, UVA, 8-oxo-dG, 8-OH-dG, 8-oxo-dGTP, Reactive oxygen species
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-66540 (URN)10.1016/j.mrfmmm.2011.07.005 (DOI)000295664200003 ()
Note

authorCount :8

Available from: 2011-12-27 Created: 2011-12-20 Last updated: 2017-12-08Bibliographically approved
2. Analysis of mutant frequencies and mutation spectra in hMTH1 knockdown TK6 cells exposed to UV radiation
Open this publication in new window or tab >>Analysis of mutant frequencies and mutation spectra in hMTH1 knockdown TK6 cells exposed to UV radiation
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2013 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 751, 8-14 p.Article in journal (Refereed) Published
Abstract [en]

Ultraviolet radiation is a highly mutagenic agent that damages the DNA by the formation of mutagenic photoproducts at dipyrimidine sites and by oxidative DNA damages via reactive oxygen species (ROS). ROS can also give rise to mutations via oxidation of dNTPs in the nucleotide pool, e.g. 8-oxo-dGTP and 2-OH-dATP and subsequent incorporation during DNA replication. Here we show that expression of human MutT homolog 1 (hMTH1) which sanitizes the nucleotide pool by dephosphorylating oxidized dNTPs, protects against mutagenesis induced by long wave UVA light and by UVB light but not by short wave UVC light. Mutational spectra analyses of UVA-induced mutations at the endogenous Thymidine kinase gene in human lymphoblastoid cells revealed that hMTH1 mainly protects cells from transitions at GC and AT base pairs.

Keyword
Ultraviolet radiation, Oxidative stress, Nucleotide pool, hMTH1, 8-Oxo-dG, 2-OH-dA
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-100126 (URN)10.1016/j.mrfmmm.2013.10.001 (DOI)000328874500002 ()
Note

AuthorCount:9;

Available from: 2014-01-27 Created: 2014-01-27 Last updated: 2017-12-06Bibliographically approved
3. Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation
Open this publication in new window or tab >>Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation
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2014 (English)In: Radiation and Environmental Biophysics, ISSN 0301-634X, E-ISSN 1432-2099, Vol. 53, no 2, 417-425 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the present study was to analyse the dose rate effect of gamma radiation at the level of mutations, chromosomal aberrations, and cell growth in TK6 cells with normal as well as reduced levels of hMTH1 protein. TK6 cells were exposed to gamma radiation at dose rates ranging from 1.4 to 30.0 mGy/h (chronic exposure) as well as 24 Gy/h (acute exposure). Cell growth, frequency of thymidine kinase mutants, and of chromosomal aberrations in painted chromosomes 2, 8, and 14 were analysed. A decline in cell growth and an increase in unstable-type chromosomal aberrations with increasing dose rate were observed in both cell lines. A dose rate effect was not seen on mutations or stable-type chromosomal aberrations in any of the two cell lines. Reduction in the hMTH1 protein does not influence the sensitivity of TK6 cells to gamma radiation. This result fits well with data of others generated with the same cell line.

Keyword
Mutations, Aberrations, Oxidative stress, Gamma radiation, Dose rate effect, hMTH1
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-113327 (URN)10.1007/s00411-014-0521-1 (DOI)000334998200020 ()24549366 (PubMedID)
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2017-12-05Bibliographically approved
4. Investigation of micronucleus induction in MTH1 knockdown cells exposed to UVA, UVB, and UVC
Open this publication in new window or tab >>Investigation of micronucleus induction in MTH1 knockdown cells exposed to UVA, UVB, and UVC
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-113334 (URN)
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2016-01-29Bibliographically approved

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